**4.3. NPs targeting the lymph node for delivering cancer vaccines**

Pattern recognition receptors (PRRs) are germline-encoded host sensors expressed mainly by cells of the innate immune system, such as MPs, (MPs) macrophages, monocytes, neutrophils, and epithelial cells, capable of detecting two classes of molecules: pathogen-associated molecular patterns (PAMPs), which are associated with microbial pathogens, and damageassociated molecular patterns (DAMPs), which are associated with components of host's cells that are released during cell damage or death [52]. PRRs play a crucial role in the proper function of the innate immune system evolved before other parts of the immune system, particularly before adaptive immunity, and mediate the initiation of Ag-specific adaptive immune response and release of inflammatory cytokines when they are activated by PRRas (PRR agonists), which are the microbe-specific molecules, including bacterial carbohydrates such as lipopolysaccharide (LPS) and mannose, bacterial peptides such as flagellin and microtubule elongation factors, peptidoglycans and lipoteichoic acids, nucleic acids such as bacterial or viral DNA and RNA, fungal glucans, and also chitin and thus are often used as vaccine adjuvants.

Since a fraction of PRRas such as LPS and unmethylated CpG ODN are soluble, prevention of rapid diffusion of free PRRas into the systemic blood circulation is indispensable for efficient targeting to professional APCs, which may be well obtained through formulating into the NP-based VADSs. This has been accomplished by dextran-CpG-OVA conjugate that enhanced not only the CD8+ T cell responses but also improved the antitumor immunotherapy through whole tumor cell vaccine. Recently, Liu and coworkers using reductive amination method conjugated oxidized dextran to amine-modified CpG ODN and demonstrated that the dextran-CpG conjugate with a hydrodynamic diameter of 6.5 nm was accumulated dLNs and was efficiently taken up by mouse DCs [53]. With the combination of OVA as a model Ag, dextran-CpG conjugate elicited production of Ag-specific CD8+ T cells for effective therapeutic benefits and in subcutaneously immunized mice resulted in significant reduction of tumor growth and increased survival of mice.

improved the survival of mice compared to the untreated group. Moon's group engineered synthetic high density lipoprotein (sHDL) nanodiscs consisting of phospholipids, apolipoprotein A1 (Apo A1)-mimetic peptides and cholesterol-conjugated CpG (sHDL-Ag/CpG) with average diameter of 10 ± 0.5 nm, which were used as a multifunctional VADS able to target lymphoid organs, resulting in sustained Ag-presenting on DCs [48]. Moreover, the sHDL-CpG-based VADS loaded with multiple Ags (MHC-I-restricted M27, MHC-II-restricted M30, and TRP2) in combination with anti-PD1/anti-CTLA4 antibodies successfully rejected B16-

Though targeting delivery with NPs is able to improve efficacy of cancer vaccines, tumorinduced DC dysfunction arising from hyperactivity of signal transducer and activator of transcription 3 (STAT3) [49], which leads to less maturation in DCs with low responsiveness to pattern recognition receptor agonist (PRRa) stimulation [50], engenders another major huddle to developing effective vaccines for cancer immunotherapy. The NPs-based VADS was trialed in overcoming tumor-induced DCs dysfunction by Ma and colleagues through using poly(ethylene glycol)-b-poly(L-lysine)-b-poly(L-leucine) (PEG-PLL-PLLeu) to form 120 nm-sized polypeptide micelles for encapsulation of polyI:C, STAT3 siRNA, and OVA as a nanovaccine (PMP/OVA/siRNA), which proved able to decrease STAT3 expression and increase CD86 and CD40 expression as well as IL-12 production [51]. Moreover, PMP/OVA/siRNA nanovaccine could effectively increase mature DCs and decrease immunosuppressive cells in tumor draining lymph node, leading to antitumor immune response and prolonged survival, implying that novel VADSs designed for codelivery of immunopotentiator and immunosuppressive gene silencer may be one of potent strategies to improve antitumor immunity by modulating tumor-induced DCs in

Pattern recognition receptors (PRRs) are germline-encoded host sensors expressed mainly by cells of the innate immune system, such as MPs, (MPs) macrophages, monocytes, neutrophils, and epithelial cells, capable of detecting two classes of molecules: pathogen-associated molecular patterns (PAMPs), which are associated with microbial pathogens, and damageassociated molecular patterns (DAMPs), which are associated with components of host's cells that are released during cell damage or death [52]. PRRs play a crucial role in the proper function of the innate immune system evolved before other parts of the immune system, particularly before adaptive immunity, and mediate the initiation of Ag-specific adaptive immune response and release of inflammatory cytokines when they are activated by PRRas (PRR agonists), which are the microbe-specific molecules, including bacterial carbohydrates such as lipopolysaccharide (LPS) and mannose, bacterial peptides such as flagellin and microtubule elongation factors, peptidoglycans and lipoteichoic acids, nucleic acids such as bacterial or viral DNA and RNA, fungal glucans, and also chitin and thus are often used as

Since a fraction of PRRas such as LPS and unmethylated CpG ODN are soluble, prevention of rapid diffusion of free PRRas into the systemic blood circulation is indispensable

F10 tumor from tumor-bearing mice.

54 Immunization - Vaccine Adjuvant Delivery System and Strategies

tumor microenvironment.

vaccine adjuvants.

**4.3. NPs targeting the lymph node for delivering cancer vaccines**

To induce a potent MHC-I-restricted CTL response which is an essential component of the successful cancer immunotherapy treatment, Huang's group formulated the mannosylated lipid-calcium-phosphate (MLCP) NPs as a new class of intracellular delivery systems for cytosol delivery into DCs of an exogenous Ag, p-Trp2 (the melanoma Ag Trp2 peptide derivative bearing two phosphor-serine residues) [54]. Compared with free Trp2 peptide/ CpG ODN, MLCP NPs encapsulation enhanced and prolonged the cargo deposit into the lymph nodes (LNs) and also resulted in superior inhibition of tumor growth in both B16F10 subcutaneous and lung metastasis mouse models owing to induced IFN-γ production and a Trp2-specific CTL immune response. Thus, encapsulation of phospho-peptide Ags into LCP may be a promising strategy for enhancing the immunogenicity of poorly immunogenic self-Ags for cancer therapy.

Recently, a nanovaccine, called AlbiVax that is assembled in vivo from endogenous albumin nanocarriers and exogenous molecular vaccines, which are chemically defined and relatively well suited to large-scale production including quality control and safety evaluation, has been developed based on the albumin properties which are well known of not only being efficiently internalized by APCs via endocytosis to facilitate intracellular vaccine delivery for optimal Ag processing and presentation but also binding to a clinically practiced Evans blue (EB) [55]. AlbiVax was synthesized by conjugating thiol-modified vaccines and adjuvants, such as the 3′-end thiol-modified CpG and Ags (CSIINFEKL, Trp2, and Adpgk) modified with N-terminal cysteine, with maleimide-functionalized EB derivative which can tightly bind to human serum albumin. Further investigation revealed that, compared to benchmark incomplete Freund's adjuvant (IFA), AlbiVax had a much high efficiency in co-delivery of CpG and Ags to LNs and in eliciting peripheral Ag-specific CD8+ CTLs with immune memory and specifically inhibited progression of established primary or metastatic EG7.OVA, B16F10, and MC38 tumors; but only in combination with anti-PD-1 and/or Abraxane did AlbiVax eradicate most MC38 tumors. These outcomes indicate that as a novel type of VADS, the in vivo self-assembled molecular nanovaccines can not only enhance vaccine bioavailability in LNs but also bypass the complications, such as inefficient delivery, sequestering Ag determinant-specific T cells in the depots, and exhausting and depleting T cells, thereby preventing T cells from infiltrating tumors and difficulty in large-scale production, which are often associated with conventional synthetic vaccines [56].

#### **4.4. NPs in combination for delivering cancer vaccines**

The VADs based on various NPs that are engineered with different adjuvants such as PRRas and tumor Ags in combination for delivery of vaccines to the same immune cells have a great potential in provoking immune responses, generating increased duration and speed of immune response, regulating Ag-antibody response, and amplifying immunogenicity of weak Ags [12]. For example, it is reported that the application of poly(γ-glutamic acid)-based NPs for the delivery of model Ag (OVA) and toll-like receptor 3 (TLR3) agonist poly (I:C) (polyinosinic-polycytidylic acid) in targeting the LNs significantly enhanced the antitumor immunity against EG7-OVA (EL-4 thymoma cells transfected with chicken albumin cDNA) in tumor-bearing mice [57]. Recently, Molino et al. designed a biomimetic approach for eliciting antitumor responses through engineering the viral-mimicking protein NP vaccine, which is pyruvate dehydrogenase E2 protein NP (of 50 nm) conjugated to gp100 epitope (melanomaassociated Ag) and CpG [58]. The CpG-gp-E2 NPs remarkably increased the proliferation of Ag-specific CD8+ T cells and production of IFN-γ and dramatically enhanced the population of CD8+ T cells in dLNs, resulting in the delayed onset of tumor growth in mice as well as elevated mouse survival, compared to control PBS-treated animals.

5 agonist (TLR5a). Unfortunately, previous clinical trials on DNA cancer vaccines, majority of which were administered as naked DNA via the intramuscular route, showed generally poor response rates, despite employment of viral vectors and electroporation able to improve the transfection of DNA vaccines, both of which cause safety and compliance concerns [61, 62]. Alternatively, NPs engineered as a VADS for intracellular delivery of DNA and mRNA provide a promising strategy for developing nucleotide-based cancer vaccines and possess several advantages [6]: (1) synthetic material-constituted NPs are safer than viral vectors, (2) NPs can stabilize and protect gene therapeutics from nuclease-mediated degradation [63], (3) DNA- and RNA-loaded NPs can be administered by injection-free tools, such as microneedles for non-parenteral delivery [64], and (4) nanocarriers can be easily modified with targeting moieties, for example, mannose, to achieve DC-targeted delivery and transfection [14, 65].

Vaccines Developed for Cancer Immunotherapy http://dx.doi.org/10.5772/intechopen.80889 57

It is reported that cationic liposomes and lipid nanoparticles containing mRNA coding for the tumor-associated Ags gp100 and TRP2 could induce a strong CD8+ T cell activation after a single immunization and treatment of B16F10 melanoma tumors with the mRNAcarried cationic liposomes resulted in tumor shrinkage and extended the overall survival of the treated mice, all of which could be further increased by the combinatory incorporation of the adjuvant LPS, showing the cationic liposomes a promising vector for mRNA vaccine delivery that is capable of inducing a strong cytotoxic T cell response for cancer immunotherapy [66]. Nevertheless, nucleotide-based vaccines, including DNA and mRNA vaccines with their intracellular Ag synthesis, have been shown to be potent activators of a cytotoxic T cell response which is an important prerequisite for successful immunotherapy against many viral diseases and tumors [67], though intracellular delivery of mRNA vaccines to the cytosol of APCs is still not sufficiently well understood and remains somewhat a challenge to clinical

With great advances in immunology and oncology, several mechanisms, involving multiple immune components, have been identified to contribute to tumor immune escape, as summarized by Chabanon and coauthors as these including [69]: (1) reduction of MHC-I molecule expression in malignant cells, resulting in decreased antigen presentation and consequently reduced detection by CTLs; (2) induction of immune cell apoptosis by cancer cells through the expression of death signals; (3) release of a variety of immune-modulatory molecules such as IL6 and IL10 by tumor cells in the microenvironment to induce immunosuppressive Tregs while inhibiting the activity of CTLs; (4) secretion of TGF-β, COX-2 (cyclooxygenase-2), and PGE2 (prostaglandin E2) by tumor cells inhibiting DC differentiation and maturation while favoring the establishment of an immunosuppressive tumor microenvironment; (5) upregulated expression of immune checkpoint ligands to activate immune checkpoint receptors providing co-inhibitory signals to CD4+ and CD8+ T cells preventing them from building a

Among these elements involved in cancer resistance, immune checkpoints are regulators of the immune system to provide pathways crucial for self-tolerance and thus play an important

translation for cancer immunotherapy [68].

specific antitumor immune response.

**6. NPs for overcoming immunosuppression**

It should be pointed out that delivery of vaccine Ags and adjuvants to target tissues or cells by a VADS is also, to a great extent, dictated by NP properties, such as particle size and surface charge, which may be appropriately engineered for improving their delivery efficiency [59]. For example, the NP-based vaccines could be either delivered actively to the lymph nodes by DCs in target tissues or transported by the interstitial flow into the lymphatics, depending mainly on NP size and surface properties such as PEGylation and charge due to the upper limit of pore size of the lymphatic capillaries and cell uptake of NPs relevant to surface properties of both [12, 60]. Wang's group engineered two types of multifunctional liposomes, the mannosylated lipid A-liposomes (MLLs) with a size of 200 nm and the stealth lipid A-liposomes (SLLs) of 50 nm, both of which were loaded with a model Ag and NH<sup>4</sup> HCO<sup>3</sup> and fabricated into microneedles, forming the proSLL/MLL-constituted microneedle array (proSMMA) as a multifunctional VADS [12]. Mice vaccinated with proSMMAs by vaginal mucosa patching administration established robust Ag-specific humoral and cellular immunity at both systemic and mucosal levels, especially, in the reproductive and intestinal ducts, under the revealed mechanism that the MLLs reconstituted from the administered microneedles were mostly taken up by vaginal mucosa resident DCs, whereas the recovered SLLs trafficked directly to dLNs wherein they are to be picked up by macrophages, proving the size of NPs as an important parameter in controlling the in vivo fate of the delivered vaccines.
